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Method for fish mononuclear/phagocyte separation, cultivation and functional verification

A technology of phagocytic cells and monocytes, used in cell culture active agents, blood/immune system cells, animal cells, etc.

Pending Publication Date: 2017-02-22
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods alone are prone to produce mixed suspensions of lymphocytes, granulocytes, platelets, and various red blood cells.

Method used

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  • Method for fish mononuclear/phagocyte separation, cultivation and functional verification
  • Method for fish mononuclear/phagocyte separation, cultivation and functional verification
  • Method for fish mononuclear/phagocyte separation, cultivation and functional verification

Examples

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Comparison scheme
Effect test

Embodiment

[0046] Embodiment: Taking tongue sole half-smooth as an example, a method for culturing, functional verification and transformation of fish phagocytes according to the present invention is introduced.

[0047] 1. Isolation and culture of peripheral blood mononuclear cells from tongue sole

[0048]Semi-smooth tongue sole was anesthetized in ice water, the back tail vein was wiped with 70% alcohol cotton ball, 2ml of peripheral blood was extracted from a heparin anticoagulant tube, and the whole blood was diluted and mixed with Hank's buffer 1:2.5-1:3 in an ultra-clean workbench, and the specific gravity was taken Put 3ml of lymphocyte separation solution between 1.05-1.09 in a 15ml centrifuge tube, add 2ml of diluted blood, centrifuge at 1800-2100rpm in a horizontal centrifuge for 18-21 minutes, collect the mononuclear cell layer, add 4-5 times the volume of Hank's buffer Wash twice, collect cell pellet by centrifugation at 900-1100rpm for 5min, resuspend in DMEM complete mediu...

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Abstract

The invention provides a method for fish mononuclear / phagocyte separation, cultivation and functional verification. Fish phagocytic cells are successfully obtained through optimizing cultivation parameters and conditions; functions of the fish phagocytic cells are obtained by utilizing the phagocytic cells for phagocytizing exogenous chicken erythrocyte and detecting in-vitro respiratory burst. The method provided by the invention realizes in-vitro separation, conversion, continuous cultivation and the like of the fishphagocytic cells, requires few samples, is short in operation time, simple in flow and high in accuracy, and can be widely applied in the research of fish immune-related functions on a cellular level.

Description

technical field [0001] The invention belongs to the technical field of biological cell culture, and in particular relates to a method for the separation, cultivation and function verification of fish mononuclear / phagocytic cells. Background technique [0002] With the expansion of aquaculture scale in recent years, the disease problems caused by harmful bacteria such as Vibrio anguillarum have seriously hindered the development of high economic value fish (such as semi-smooth tongue sole) aquaculture industry. At present, there is no effective immune control technology and method, and traditional methods such as chemical drugs and antibiotics are still used to control the occurrence of diseases. Peripheral blood mononuclear cells (precursors of phagocytes) are an important part of fish humoral immunity and play an important role in the process of fish specific immunity and nonspecific immunity. At present, the research on the immune function of monocytes in tongue sole and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786A61K49/00
CPCA61K49/0008C12N5/0645C12N2501/115Y02A40/81
Inventor 沙珍霞陈亚东孙璐明汪林庆陈松林
Owner QINGDAO UNIV
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